The formation and release of inflammatory mediators by mast cells play central roles in immediate hypersensitivity reactions and contribute to the development and perpetuation of chronic inflammation in diseases such as asthma. The long range goal of this work is to understand what roles lipid metabolic pathways play in specific subcellular compartments in causing and/or regulating mast cell mediator release. Attention is focused on 1,2-diacylglycerol (DAG) - a lipid intermediate that increases 2-5 fold with physiologic stimulation, participates in the regulation of phosphorylation of proteins by protein kinase C and acts as a substrate both for the release of arachidonic acid and the formation of fusogenic lipids potentially important in exocytosis. A new conceptual approach to study the mechanism(s) of DAG formation and consumption has been developed. The molecular species "fingerprint" of DAG was determined and found to be discordant with that of phosphatidylinositol (the lipid felt by most investigators to give rise to the increases DAG mass occurring in cellular activation) strongly suggesting that mast cell DAG is principally formed by other pathways. This observation and others strongly motivate a reassessment of the mechanisms that form and remove DAG in specific subcellular sites. Aim 1 proposes to define the relative importance, location and regulation of different DAG generative pathways by 1a) Employing deductive analysis after determining the molecular species fingerprints of putative precursor phospholipids and triglyceride and 1b) Complementary studies examining in vitro the activity, subcellular location and regulation of specific enzymes of interest. Because the mess of any intermediate is controlled not only by the rate of its formation, but equally importantly by its rate of removal, Aim II seeks to identify the relative importance of specific DAG metabolic pathways, their location and regulation. Four approaches this question include: 2a) Molecular species analysis of DAG metabolites, 2b) Studies of the metabolism of DAG generated in situ or added exogenously, 2c) Determining the mass of putative DAG metabolites, and 2d) direct assessment in vitro of appropriate enzymes. These studies of the nature and subcellular location of DAG metabolism -a process integral to the biochemical mechanisms regulating mediator release - should contribute to a much clearer view of immunologic activation mast cells. This knowledge will help to facilitate the development of new pharmacologic approaches to allergic disorders.